Advisory Committee on Health Effects of Endocrine Disruptors
The Supplement II to the Intermediary Report
1.2.4.2

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Analysis of phthalic acid esters in animal feeds

[Outline]
Phthalic acid esters (PAEs)^{1, 2}, extracted from feeds for experimental animals with acetonitrile and purified by Florisil-PSA column chromatography, are analyzed by a gas chromatograph/mass spectrometer (GC/MS) qualitatively and quantitatively.
The esters analyzed here are dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP), diisooctyl phthalate (DiOP) and diisononyl phthalate (DiNP).
This method can be applied to feeds³.

[Reagents]
Confirm by chromatography that all the reagents present no problem in PAE analysis.
(i) Organic solvents: n-Hexane, acetonitrile and acetone of PAE analysis grade. Open the seal immediately before use⁴.
(ii) Anhydrous odium sulfate: Residual pesticide analysis grade; heat to 200℃ for 2 hours and let cool down before use.
(iii) Sodium chloride: PAE analysis grade; heat to 200℃ for 2 hours and let cool down before use.
(iv) Purified water: Distilled water after washing with n-hexane of the PAE analysis grade⁵.
(v) Florisil: Florisil PR; heat to 200℃ for 2 hours and let cool down before use.
(vi) PSA: Bondesil PSA.
(vii) Standard PAE solutions: Place 20 mg each of standard products in 20 ml measuring flasks and add n-hexane to 20 ml to obtain 1000 μg/ml solutions. Dilute as appropriate.
(viii) Internal standards: Place 20 mg each of standard PAE-d4 in 20 ml measuring flasks and add n-hexane to 20 ml to obtain 1000 μg/ml solutions. Dilute as appropriate.

[Apparatus]
(i) Gas chromatograph/mass spectrometer (GC/MS): A unit capable of selective ion monitoring (SIM) equipped with an ion source by electron ionization and a capillary column (splitless)
(ii) Florisil-PSA column: Fill a glass chromatographic column, 15 mm i.d. and 110 mm long, with 1 g Florisil and 0.5 g PSA and 2 g anhydrous sodium sulfate upon it. Wash the column with 10 ml acetone and 10 ml n-hexane before use.

[Preparation of sample solutions]⁶
Place 5 g feed in a 50 ml stoppered glass centrifuge tube. Add 5 ml purified water, 5 ml acetonitrile, and 125 μl internal standard solution (4 μg/ml), and homogenize for 1 min. After centrifuging the solution for 5 min at 3000 rpm, separate the supernatant. Add 5 ml purified water and 15 ml acetonitrile to the residue and perform the same operation. Merge the supernatants, add 1.5 g sodium chloride and shake for 5 min.
Separate the acetonitrile layer, add 4 ml of n-hexane saturated with acetonitrile and shake for 5 min. Separate the acetonitrile layer, and remove acetonitrile by distillation under reduced pressure. Dissolve the residue in 2 ml purified water, add 5 ml n-hexane, and transfer to a 20-ml stoppered glass test tube. After shaking for 30 seconds, centrifuge for 5 min at 3000 rpm, and separate the n-hexane layer. Add 5 ml n-hexane to the water layer, and perform the same operation. Charge the Florisil-PSA column with the n-hexane layer, wash the column with 3 ml n-hexane, and elute PAE with 10 ml of 5% acetone-n-hexane. Concentrate the eluent to dryness under reduced pressure, and add n-hexane to 1 ml.
　

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